Potentiometric sensor and analytical element

ABSTRACT

A potentiometric sensor with suppressed leak current on the surface of an electrode and improved for a dynamic range and a response speed, in which a redox compound is immobilized through insulative molecules on the surface of a gold electrode, and a current between a source and drain of an insulated gate field-effect transistor along with reaction between an oxidized substance or a reduced substance produced by the reaction of a measured substance in a sample solution injector for supplying the sample solution containing the measured substance and an enzyme and a redox compound on the surface of the gold electrode, is monitored on real time to measure the change of the surface potential.

CLAIM OF PRIORITY

The present application claims priority from Japanese application JP2006-313876 filed on Nov. 21, 2006, the content of which is herebyincorporated by reference into this application.

This application is a Divisional application of application Ser. No.11/942,862, filed Nov. 20, 2007, now U.S. Pat. No. 8,202,409, thecontents of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention concerns a sensor and an analytical elementcapable of measuring a biological substance at a high sensitivity bysubjecting a biological substance selectively to redox reaction using anenzyme or the like and measuring the surface potential formed therein.

BACKGROUND OF THE INVENTION

Blood test in health check is effective for recognizing the state ofheath and early detection of a disease. In the blood test upon healthcheck, since a large number of samples are analyzed over multipletargets, large-scaled clinical analyzers are used. Since the clinicalanalyzers are expensive and have to be operated by expert engineers,they are introduced in large-scaled hospitals or blood testing centers,but are not placed in general clinics. Accordingly, when blood test isperformed in a general clinic, it usually takes several days forobtaining the result. This time lag causes no problem in the case ofhealth checks since most of them are performed at a frequency of onceper year or one half year. In an urgent case, however, it is necessaryto conduct blood test on the spot. For example, during surgery, it isnecessary to monitor blood electrolytes such as sodium, potassium, orchlorine, an oxygen partial pressure, a carbon dioxide gas partialpressure, glucose, blood urea nitrogen, hematocrit. Further, in dialysisfor renal insufficiency, creatinine is measured. In addition to suchurgent testing, it is also a demand for point of care testing (POCT) inorder to check the health state in general clinics. The apparatus copingwith such demand is a POCT apparatus, which has an advantage capable oftesting on-site optionally although the number of test targets andthroughputs are not so favorable as those of the clinical analyzer. Thetest targets include electrolyte, glucose, cholesterol, lactic acid,blood urea nitrogen, and creatinine. For general chemical measurementfor glucose, cholesterol, lactic acid, blood urea nitrogen, andcreatinine other than the electrolyte, an enzyme electrode method isused.

The enzyme electrode method is a method of measuring the concentrationof the substance, which is converted into another substance capable ofbeing measured by an electrode using an enzymatic reaction, indirectlyby the electrode as a current or potential change. For example, in aglucose sensor for measuring a blood glucose level, glucose as asubstance to be measured is oxidized by a glucose oxidase andgluconolactone is produced. By the oxidation reaction, oxygen isconsumed to produce hydrogen peroxide. Since both oxygen and hydrogenperoxide are redox active compounds, the concentration of the glucose asthe substance to be measured can be measured by using an oxygenelectrode or a hydrogen peroxide electrode as the electrode current.However, in a case where the glucose is at a high concentration, therate of oxidation reaction is sometimes limited by the concentration ofdissolved (partial pressure) oxygen in the blood. As a countermeasure,other redox compound is sometimes used instead of oxygen. Other chemicalsubstances can also be measured on a similar principle. Such a type ofsensor is generally referred to as an amperometric enzyme sensor.

In the amperometric enzyme sensor, a working electrode, formed of gold,platinum or the like, a counter electrode and a reference electrode forkeeping the potential of the working electrode constant are arranged ina solution, and an enzyme and a redox compound are in the solution. Theworking electrode, the counter electrode, and the reference electrodeare connected to a current measuring device such as a potentiostat, suchthat a current value which changes upon application of a voltage betweenthe working electrode and the counter electrode can be measured. When asample (for example, blood) containing a substance to be measured isadded to the solution, the substance is oxidized by the enzyme and, atthe same time, the redox compound in the oxidized state is reduced. Whena constant voltage capable of oxidizing the redox compound is applied tothe working electrode, the redox compound in the reduced state isoxidized on the working electrode and a current flows in accordance withthe concentration of the redox compound in the reduced state. In thisway, the oxidation reaction of the substance to be measured by theenzyme can be measured as a current, and the concentration of thesubstance to be measured can be measured indirectly. In this case, it isnecessary for an enzyme at a sufficient concentration, a redox compoundat a sufficient concentration, and a working electrode of a sufficientsize such that a current value in accordance with the concentration ofthe substance to be measured can be obtained, that is, the concentrationof the substance to be measured is a rate determining factor in thereaction system.

In the amperometric enzyme sensor, an enzyme is immobilized on amembrane mainly with an aim of re-utilizing the enzyme. However, in acase where the enzyme is immobilized, since the reaction efficiency ofthe enzyme and the substance to be measured and the enzyme and the redoxcompound is lowered, the redox compound is immobilized together with theenzyme on the membrane at the surface of the working electrode (Adv.Mater. 5(1993) 912-915). It is considered that lowering of the transferefficiency of charges from the enzyme to the redox compound can besuppressed by immobilizing the enzyme together with the redox compoundon the membrane at the surface of the electrode. Further, byimmobilizing the enzyme and the redox compound at a multilayer, thesensitivity is improved more and lowering of the reaction efficiencybetween the enzyme and the object to be measured can be suppressedcompared with a case of a monolayer.

In the glucose sensor for measuring the blood glucose level, since anecessary measuring sensitivity is not so high, measuring is possiblewith a blood amount of several droplets. However, in a POCT apparatusfor general targets, more amount of blood is necessary for maintainingthe measuring sensitivity. For example, i-Stat developed as a POCTapparatus (Clin. Chem. 39/2 (1993) 283-287) required a blood amount ofabout 65 μl. While the blood amount can be decreased by making theelectrode area smaller, since a signal (that is, current value)decreases as the electrode area is made smaller in the amperometricenzyme sensor, it was difficult to simply decrease the electrode area.

A potentiometric enzyme sensor is known as an enzyme sensor using anelectric measuring method in which signals do not depend on theelectrode area. The potentiometric enzyme sensor consists of a workingelectrode formed of gold, platinum, etc. and a reference electrode inwhich an enzyme and a redox compound are present in the measuringsolution (JP-T No. 9-500727). Further, the working electrode and thereference electrode are connected to a device for measuring voltage.When a substance to be measured is added to the measuring solution, thesubstance to be measured is oxidized by an enzymatic reaction and, atthe same time, a redox compound in an oxidized state is reduced. Thesurface potential on the working electrode generated in this case is inaccordance with the following Nernst's equation.

$\begin{matrix}{E = {E^{0} + {\frac{RT}{n\; F}{\ln( {C_{ox}/C_{red}} )}}}} & \lbrack {{Formula}\mspace{14mu} 1} \rbrack\end{matrix}$whereE: surface potential of working electrode,E⁰: reference potential of redox compoundR: gas constantT: absolute temperaturen: difference of charges between oxidized state and reduced state ofredox compoundF: Faraday constantC_(ox): concentration of oxidized state of redox compoundC_(red): concentration of reduced state of redox compound

As can be seen from the equation described above, the change of thesurface potential does not depend on the electrode area. Further, unlikethe amperometric enzyme sensor, since a voltage is not applied to theworking electrode, chemical reaction interfering the measurement lessoccurs. Further, since the voltage changes as the logarithm of theconcentration as shown in the equation described above, a substance canbe measured also in a low concentration region at an S/N ratio identicalwith that in a high concentration region and it is considered that awider dynamic range can be obtained compared with the amperometricenzyme sensor.

However, in the existent potentiometric enzyme sensor, consideration isnot taken on the insulative property between the working electrode andthe measuring solution, and it involved a problem that actualmeasurement undergoes the effect of a leak current on the surface of theelectrode and the sensitivity is lowered, particularly, in the lowconcentration region, the dynamic range narrowed and, further, theresponse speed is lowered.

SUMMARY OF THE INVENTION

The present invention intends to provide a potentiometric sensor ofsuppressing a leak current at the surface of an electrode which causes aproblem in actual measurement and improving a dynamic range and aresponse speed.

This is attained according to the present invention by immobilizing aredox compound through insulative molecules on the surface of theworking electrode not allowing the current to flow, for example, acarbon chain and measuring the change of a surface potential along withthe reaction of an oxidation substance or a reduction substance producedby the reaction between a substance to be measured and an enzyme and aredox compound on the surface of the working electrode. Further, as apotentiometric device, an insulated gate field-effect transistor formedon a substrate identical with that for the working electrode is used.

According to the invention, by immobilizing the redox compound throughthe insulative molecules on the surface of the working electrode, aworking electrode having a property responsible to an oxidized substanceor a reduced substance in a solution and having a high insulativeproperty to the solution can be obtained. Improvement in the insulativeproperty can suppress the leak current on the surface of the electrode,particularly, improve the sensitivity in the low concentration regionand can extend a dynamic range, as well as the response speed can beincreased. Further, hydrogen peroxide or a thiol compound that could notbe measured repetitively so far by the potential difference can bemeasured repetitively by the potential difference. Then, since theapparent concentration of the redox compound does not depend on theamount of the solution, measurement is possible not depending on theamount of the blood. By using an insulated gate filed-effect transistorformed on a substrate identical with that for the working electrode as apotentiometric device, the leak can be decreased, dynamic range can beextended and the response speed can be improved. Further, byimmobilizing the redox compound on the working electrode with insulativemolecules of an identical length, working electrode can obtaininsulative property and change of state of the immobilized redoxcompound can give an effect on the surface potential of the workingelectrode uniformly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing an example of a potentiometric enzymesensor according to the invention;

FIG. 2 is a block diagram showing an example of a potentiometric enzymesensor according to the invention;

FIG. 3 is a block diagram showing an example of a potentiometric enzymesensor using an FET sensor according to the invention;

FIG. 4 is a block diagram showing an example of a potentiometric enzymesensor using an FET sensor according to the invention;

FIG. 5 is a block diagram showing an example of a potentiometric enzymesensor using an EFT sensor according to the invention capable ofmeasuring plural targets together;

FIG. 6 is a view showing an example of an analytical element used for apotentiometric enzyme sensor using an FET sensor according to theinvention;

FIG. 7 is an explanatory view for an evaluation system of immobilizing aredox compound through insulative molecules on the surface of a workingelectrode;

FIG. 8 is a graph showing the effect of immobilizing a redox compoundthrough insulative molecules on the surface of a working electrode;

FIG. 9 is a graph showing the effect of immobilizing a redox compoundthrough insulative molecules on the surface of a working electrode;

FIG. 10 is an explanatory view for an evaluation system of immobilizinga redox compound through insulative molecules on the surface of aworking electrode;

FIG. 11 is a graph showing the effect of immobilizing a redox compoundthrough insulative molecules on the surface of a working electrode;

FIG. 12 is a graph showing the result of measuring hydrogen peroxideusing a working electrode on which a redox compound is immobilizedthrough insulative molecules;

FIG. 13 is a graph showing the result of measuring glucose concentrationby using a potentiometric enzyme sensor according to the invention;

FIG. 14 is a graph showing the result of measuring cholesterolconcentration by using a potentiometric enzyme sensor according to theinvention;

FIG. 15 is a graph showing the result of measuring Fru-Val-His substrateby using a potentiometric enzyme sensor according to the invention;

FIG. 16 is a graph showing the result of measuring cholinesteraseactivity by using a potentiometric enzyme sensor according to theinvention;

FIG. 17 is a graph showing the result of repetitively measuring a thiolcompound by using an FET sensor according to the invention;

FIG. 18 is a block diagram showing an example of an enzyme immunoassaysystem by using an FET sensor according to the invention; and

FIG. 19 is a graph showing the result of repetitively measuring IL-1β byusing an FET sensor according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is to be described by way of preferred embodimentswith reference to the drawings.

FIG. 1 is a block diagram showing an example of a potentiometric enzymesensor according to the invention. The measuring system of thisembodiment includes a measuring station 101, a signal processing circuit102, and a data processing apparatus 103. The measuring station 101 hasa potentiometric device 104, a reference electrode 105, a samplesolution injector 106 for supplying a sample solution containing asubstance to be measured, and a measuring cell 107. In the measuringsolution 111 in the measuring cell 107, there are placed an enzyme 108,a gold electrode 112 on which a redox compound 109 is immobilizedthrough insulative molecules 110 and the reference electrode 105. Whilean oxidase conducting oxidation and reduction is used as an enzyme, anyenzyme that produces an oxidized substance or reduced substance byenzymatic reaction can be used with no problem. For example,acetylcholinesterase that hydrolyzes acetylthiocholine to produces athiol compound, which is a reduced substance, can also be used.

Measuring procedures are as described below. The sample solution isinjected by using the sample solution injector 106 into the measuringsolution 111 in the measuring cell 107. A substance to be measured inthe sample solution is oxidized by the enzymatic reaction and, alongwith the reaction, dissolved oxygen in the sample solution is reducedinto hydrogen peroxide. The produced hydrogen peroxide oxidizes theredox compound 109. As a result, the potential on the gold electrode 112changes. The potential difference between the reference electrode 105and the gold electrode 112 that changes before and after the injectionof the sample solution by the sample solution injector 106 is measuredat a real time by the potentiometric device 104, and recorded by thesignal processing circuit 102 and the data processing apparatus 103. Therate of change of the potential on the gold electrode 112 depends on therate of producing hydrogen peroxide, that is, the reaction rate ofoxidation of the substance to be measured, and the reaction rate of theoxidation of the substance to be measured depends on the concentrationof the substance to be measured. Accordingly, by measuring the rate ofchange of the potential difference between the reference electrode 105and the gold electrode 112, the concentration of the substance to bemeasured can be obtained.

Like the rate of change of the potential difference between thereference electrode 105 and the gold electrode 112, the quantity ofchange of the potential difference between the reference electrode 105and the gold electrode 112 during a certain period depends on theconcentration of the measured substance. Accordingly, the concentrationof the substance to be measured can be obtained by measuring thequantity of change between the potential difference before the injectionof the sample solution or just after the injection of the samplesolution and the potential difference at a predetermined time after theinjection of the sample solution. Further, in a case where thereproducibility of the potential difference between the referenceelectrode 105 and the gold electrode 112 before the injection of thesample solution is higher than the accuracy required for measurement,the concentration of the substance to be measured can be obtained bymeasuring the potential difference at a predetermined time after theinjection of the sample solution.

Preferably, the redox compound 109 is previously in a reduced state in acase of measuring the oxidizing substance such as hydrogen peroxide. Forproviding a reduced state, it is treated with a reducing agent such aspotassium ferrocyanide, sodium thiosulfate or dithiothreitol (DTT), or areducing potential is applied by using a potentiostat. By applying thereducing treatment, the potential before the injection of the samplesolution can be stabilized and, further, the sensitivity at a lowconcentration can be improved to extend the dynamic range. In the samemanner, in a case of measuring the reducing substance such as the thiolcompound, it is preferably formed into an oxidized state. For providingthe oxidizing state, it is treated by an oxidizing agent such aspotassium ferricyanide, potassium permanganate or hydrogen peroxide, oran oxidizing potential is applied by using a potentiostat. By previouslyapplying the oxidizing treatment, the potential before the injection ofthe sample solution can be stabilized and, further, the sensitivity at alow concentration can be improved to extend the dynamic range.

For the insulative molecules 110, an alkane thiol is used preferably. Bythe use of the alkane thiol, a highly insulative monolayer can be formedeasily on the surface of a noble metal such as gold or silver. With aview point of the insulative property, the alkyl chain in thealkanethiol has a carbon chain of 6 or more.

The reference electrode 105 provides a reference potential for stablymeasuring the potential change based on an equilibrium reaction orchemical reaction occurring at the surface of the gold electrode 112 inthe measuring solution 111. A silver chloride electrode or a calomelelectrode using saturated potassium chloride as an internal solution isusually used as the reference electrode. A silver chloride electrodewithout an internal solution may be used as a quasi-reference electrode.

Instead of the gold electrode 112, an electrode comprising other noblemetal such as silver or carbon may also be used.

A sample solution is not present in the measuring solution 111 beforeinjection of the sample solution but a sample solution supplied from thesample solution injector 106 may function as such solution. In thiscase, the enzyme 108 may be previously solidified by freeze drying orthe like and dissolved by a sample solution supplied from the samplesolution injector 106.

For oxidation and reduction of the measured substance by the enzyme 108,an enzyme for the pretreatment of the measured substance or a substrateneeded for redox reaction may sometimes be used.

The enzyme 108 in accordance with the measured substance and the enzymefor the pretreatment are exemplified in Table 1.

TABLE 1 Target Enzyme Glucose Glucose oxidase Cholesterol Cholesteroloxidase (for pretreatment: cholesterol esterase) Lactate Lactate oxidaseCreatine Sarcosine oxidase (for pretreatment: creatinase) CreatinineSarcosine oxidase (for pretreatment: creatininase, creatinase) PyruvatePyruvate oxidase (substrate: phosphoric acid) Hemoglobin Fructosyl-aminoacid oxidase, or fructosyl-peptide A_(lc) oxidase (for pretreatment:protease) Alcohol Alcohol oxidase Triglyceride Glycerol oxidase (forpretreatment: Lipase)

FIG. 2 is a block diagram showing an example of a potentiometric enzymesensor according to the invention. The measuring system of thisembodiment includes a measuring station 201, a signal processing circuit202 and a data processing apparatus 203. The measuring station 201 has apotentiometric device 204, a reference electrode 205, a sample solutioninjector 206 for supplying a sample solution containing a measuredsubstance, and a measuring cell 107. In the measuring solution 212 inthe measuring cell 207, there are placed an enzyme 208, a water solubleredox compound 209 not immobilized on the gold electrode 213, a goldelectrode 213 on which a redox compound 210 is immobilized throughinsulative molecules 211, and the reference electrode 205.

The measuring procedures are as described below. A sample solution isinjected by using the sample solution injector 206 into the measuringsolution 212 in the measuring cell 207. The measured substance in thesample solution is oxidized by the enzymatic reaction and, along withthe reaction, the redox compound 209 in the sample solution is reduced.The produced redox compound 209 in the reduced state oxidizes the redoxcompound 210. As a result, the potential on the gold electrode 213changes. The potential difference between the reference electrode 205and the gold electrode 213 that changes before and after the injectionof the sample solution by the sample solution injector 206 is measuredat a real time and recorded by the signal processing circuit 202 and thedata processing apparatus 203. The rate of change of the potential onthe gold electrode 213 depends on the rate of producing the redoxcompound 209 in the reduced state, that is, the reaction rate ofoxidation of the measured substance and the reaction rate of oxidationof the measured substance depends on the concentration of the measuredsubstance. Accordingly, the concentration of the measured substance canbe obtained by measuring the rate of change of the potential differencebetween the reference electrode 205 and the gold electrode 213.

Like the rate of change of the potential difference between thereference electrode 205 and the gold electrode 213, the quantity ofchange of the potential difference between the reference electrode 205and the gold electrode 213 during a certain period depends on theconcentration of the measured substance. Accordingly, the concentrationof the measured substance can be obtained by measuring the quantity ofchange of the potential difference before the injection of the samplesolution or just after the injection of the sample solution and thepotential difference at a predetermined time after the injection of thesample solution. Further, in a case where the reproducibility of thepotential difference between the reference electrode 205 and the goldelectrode 213 before the injection of the sample solution is higher thanthe accuracy required for the measurement, the concentration of themeasured substance can be obtained by measuring the potential differenceat a predetermined time after the injection of the sample solution.

In a case of measuring the oxidation reaction of the measured substance,it is preferred that the redox compound 209 is previously in an oxidizedstate. By providing the oxidized state the sensitivity at lowconcentration can be improved to extend the dynamic range.Alternatively, it is preferred that the ratio of the oxidation andreduction state is known for the redox compound 209 and the quantity inthe reduced state is less than the quantity of the measured substance.For example, in a case where the concentration of the measured substanceis 1 mM, it is set such that the concentration of the redox compound is10 mM and oxidized state:reduced state=99:1 before the injection of themeasured substance. After the injection of the measured substance, theredox compound ratio changes to oxidized state:reduced state=89:11 inaccordance with the oxidation of the measured substance. The change ofthe ratio can be detected as the change of the surface potential inaccordance with the Nernst's equation. In the same manner, in a case ofmeasuring the reducing reaction of the measured substance, it ispreferred that the redox compound 209 is previously in a reduced state.By providing the reduced state, the sensitivity at a low concentrationcan be improved to extend the dynamic range. Alternatively, it ispreferred that the ratio of the oxidation and reduction state for theredox compound 209 is known and the quantity of the oxidized state isless than the quantity of the measured substance.

It is sometimes preferred that the redox compound 209 is a mixture ofdifferent kind of redox compounds. For example, while nicotinamideadenine dinucleotide (NAD) acts as an electron receptor in the oxidationreaction of cholesterol by a cholesterol dehydrogenase, ferricyan lessact as an electron receptor in the reaction. On the other hand, in acase of using ferrocene for the redox compound 210 immobilized on thegold electrode 213, the redox reaction less occurs between ferrocene andNAD and it is difficult to measure the reduction of NAD as the change ofpotential. However, since redox reaction is taken place betweenferrocene and ferricyan, reduction of ferricyan can be measured as thechange of potential. In this case, by the use of a mixture of NAD andferricyan as the redox compound 209, an equilibrium reaction is causedbetween NAD and ferricyan, and the reduction of NAD can be measured byway of reduction of ferricyan as the change of potential.

For the insulative molecules 211, an alkane thiol is used preferably. Bythe use of the alkane thiol, a highly insulative monolayer can be formedeasily on the surface of a noble metal such as gold or silver. With aview point of the insulative property, the alkyl chain in thealkanethiol has a carbon chain of 6 or more.

The reference electrode 205 provides a reference potential for stablymeasuring the potential change based on an equilibrium reaction orchemical reaction occurring at the surface of the gold electrode 213 inthe measuring solution 212. A silver chloride electrode or a calomelelectrode using saturated potassium chloride as an internal solution isusually used as the reference electrode. A silver chloride electrodewithout an internal solution may be used as a quasi-reference electrode.

Instead of the gold electrode 213, an electrode comprising other noblemetal such as silver or carbon may also be used.

A sample solution is not present in the measuring solution 212 beforeinjection of the sample solution but a sample solution supplied from thesample solution injector 206 may function as such solution. In thiscase, the enzyme 208 and the redox compound 209 may be previouslysolidified by freeze drying or the like and dissolved by a samplesolution supplied from the sample solution injector 206.

For oxidation and reduction of the measured substance by the enzyme 208,an enzyme for the pretreatment of the measured substance or a substrateneeded for redox reaction may sometimes be used.

As the enzyme 208 in accordance with the measured substance, thoseexemplified in Table 2 can be used in addition to those exemplified inTable 1.

TABLE 2 Target Enzyme Glucose Glucose dehydrogenase CholesterolCholesterol dehydrogenase (for pretreatment: cholesterol esterase)Lactate Lactate dehydrogenase Pyruvate Pyruvate dehydrogenase(substrate: phosphoric acid) Alcohol Alcohol dehydrogenase TriglycerideGlycerol dehydrogenase (for pretreatment: Lipase)

FIG. 3 is a block diagram showing an example of a potentiometric enzymesensor using an FET (Field Effect Transistor) sensor according to theinvention. The measuring system of this embodiment includes a measuringstation 301, a signal processing circuit 302, and a data processingapparatus 303. The measuring station 301 includes an insulated gatefield-effect transistor 304, a reference electrode 307, a power source308, a sample solution injector 309 for supplying a sample solutioncontaining a measured substance, and a measuring cell 310. The insulatedgate field-effect transistor 304 has a source 305, a drain 306, and agold electrode 315 electrically connected with a gate. In a measuringsolution 314 in the measuring cell 310, there are placed an enzyme 311,the gold electrode 315 on which a redox compound 312 is immobilizedthrough insulative molecules 315, and the reference electrode 307. Whilean oxidase for oxidation and reduction is used as the enzyme, any enzymethat produces an oxidized substance or a reduced substance by anenzymatic reaction can be used with no trouble. For example, acetylcholinesterase that hydrolyzes acetylthiocholin to produces a thiolcompound, which is a reduced substance, can also be used.

The measuring procedures are as described below. A predetermined voltageis applied from the power source 308. A sample solution is injected byusing the sample solution injector 309 into the measuring solution 314in the measuring cell 310. The measured substance in the sample solutionis oxidized by the enzymatic reaction and, along with the reaction, thedissolved oxygen in the sample solution is reduced to hydrogen peroxide.The produced hydrogen peroxide oxidizes the redox compound 312. As aresult, the potential on the gold electrode 315 changes. A DC componentof a current between the source 305 and the drain 306 in the insulatedgate field-effect transistor 304 that changes before and after theinjection of the sample solution by the sample solution injector 309 ismeasured at a real time and recorded by the signal processing circuit302 and the data processing apparatus 303. The rate of change of the DCcomponent of the current between the source 305 an the drain 306 dependson the potential on the gold electrode 315, and the potential on thegold electrode 315 depends on the rate of producing hydrogen peroxide,that is, the reaction rate of oxidation of the measured substance andthe reaction rate of the oxidation of the measured substance depends onthe concentration of the measured substance. Accordingly, theconcentration of the measured substance can be obtained by measuring therate of change of the DC component of the current between the source 305and the drain 306.

Like the rate of change of the DC component of the current between thesource 305 and the drain 306, the quantity of change of the DC componentof the current between the source 305 and the drain 306 in apredetermined period depends on the concentration of the measuredsubstance. Accordingly, the concentration of the measured substance canbe obtained by measuring the quantity of change of the DC component ofthe current between the source 305 and the drain 306 before injection ofthe sample solution or just after the injection of the sample solution,and at a predetermined period after the injection of the samplesolution. Further, in a case where the reproducibility of the DCcomponent of the current between the source 305 and the drain 306 beforeinjection of the sample solution is higher than the accuracy requiredfor the measurement, the concentration of the measured substance can beobtained by measuring the DC component of the current between the source305 and the drain 306 at a predetermined time after the injection of thesample solution.

It is preferred that the redox compound 312 is previously in a reducedstate in a case of measuring an oxidation substance such as hydrogenperoxide. For providing the reduced state, the substance is treated witha reducing agent such as potassium ferrocyanide, sodium thiosulfate, orDithiothreitol (DTT). By providing the reduced state, it is possible tostabilize the potential before the injection of the sample solution and,further, improve the sensitivity at a low concentration to extend adynamic range. In the same manner, in a case of measuring a reducingsubstance such as a thiol compound, it is preferred that the substanceis previously in an oxidized state. For providing an oxidized state, thesubstance is treated with an oxidizing agent such as potassiumferricyanide, potassium permanganate, or hydrogen peroxide. By applyingthe oxidizing treatment, it is possible to stabilize the potentialbefore injection of the sample solution and, further, improve thesensitivity at a low concentration to extend a dynamic range.

In a case where the measured substance is oxidized or reduced by theenzyme 311 and, as a result, the oxidation and reduction state of theredox compound 312 changes, the oxidation and reduction state of theredox compound 312 may be changed by way of hydrogen peroxide asdescribed above, by way of another redox compound as shown in FIG. 2, ordirectly by the oxidation reduction for the measured substance not byway of any redox compound.

For the insulative molecules 313, an alkanethiol is used preferably. Bythe use of the alkanethiol, a highly insulating monolayer can be formedeasily on the surface of a noble metal such as gold or silver. With aview point of the insulative property, the alkyl chain in thealkanethiol has a carbon chain of 6 or more.

The reference electrode 307 provides a reference potential for stablymeasuring the potential change based on the equilibrium reaction orchemical reaction occurring at the surface of the gold electrode 315 inthe measuring solution 314. A silver chloride electrode or calomelelectrode using saturated potassium chloride as an internal solution isusually used as the reference electrode. A silver chloride electrodewithout an internal solution may be used as a quasi-reference electrode.

Instead of the gold electrode 315, an electrode formed of other noblemetal such as silver may also be used.

A sample solution is not present in the measuring solution 314 beforeinjection of the sample solution but a sample solution supplied from thesample solution injector 309 may function as such solution. In thiscase, the enzyme 311 may be previously solidified by freeze drying orthe like and dissolved by a sample solution supplied from the samplesolution injector 309.

The power source 308 is preferably a power source containing an ACcomponent. By superimposing an AC voltage at 1 KHz or higher to the DCcomponent, stabilization of the surface potential of the gold electrode315 can be expected.

For oxidation and reduction of the measured substance by the enzyme 311,an enzyme for the pretreatment of the measured substance or a substrateneeded for redox reaction may sometimes be used.

As the enzyme 311 in accordance with the measured substance, thoseexemplified in Table 1 and Table 2 can be used.

FIG. 4 is a block diagram showing an example of a potentiometric enzymesensor using an FET sensor according to the invention. The measuringsystem of this embodiment includes a measuring station 401, a signalprocessing circuit 402, and a data processing apparatus 403. Themeasuring station 401 includes an insulated gate field-effect transistor404, a power source 407, a quasi-reference electrode 408, a samplesolution injector 410 for supplying a sample solution containing ameasured substance and a measuring cell 411. The insulated gatefield-effect transistor 404 has a source 405, a drain 406, and a goldelectrode 417 electrically connected with a gate. In the measuring cell411, there are placed a blood cell separation membrane 412, ainterfering substance separation membrane 413, an enzyme 414, a goldelectrode 417 on which a redox compound 415 is immobilized through theinsulative molecules 415, and the quasi-reference electrode 408, and agel 409 containing a reference potential solution. While an oxidase foroxidation and reduction is used as the enzyme, any enzyme that producesan oxidized substance or a reduced substance by an enzymatic reactioncan be used with no trouble. For example, acetyl cholinesterase thathydrolyzes acetylthiocholin to produces a thiol compound, which is areduced substance, can also be used.

The measuring procedures are as described below. A predetermined voltageis applied from the power source 407. A sample solution such as a bloodis injected by using the sample solution injector 410 in the measuringcell 411. The blood cell in the sample solution is removed by the bloodcell separation membrane 412 and interfering substances giving an effecton the surface potential of ascorbic acid, etc. in the sample solutionare removed by the interfering substance separation membrane 413. Theliquid permeating the separation membranes dissolves the enzyme 414. Themeasured substance in the sample solution is oxidized by the enzymaticreaction and, along with the reaction, dissolved oxygen in the samplesolution is reduced to hydrogen peroxide. The produced hydrogen peroxideoxidizes the redox compound 415. As a result, the potential on the goldelectrode 417 changes. A DC component of a current between the source405 and the drain 406 in the insulated gate field-effect transistor 404is measured at a real time and recorded by the signal processing circuit402 and the data processing apparatus 403. The rate of change of the DCcomponent of the current between the source 405 and the drain 406depends on the potential of the gold electrode 417, and the potential onthe gold electrode 417 depends on the rate of producing hydrogenperoxide, that is, the reaction rate of oxidation of the measuredsubstance and the reaction rate of the oxidation of the measuredsubstance depends on the concentration of the measured substance.Accordingly, the concentration of the measured substance can be obtainedby measuring the rate of change of the DC component of the currentbetween the source 405 and the drain 406.

In a case of using urine as the sample solution, the urine is preferablydiluted and injected into the measuring cell 411 by using the samplesolution injector 410. In this case, the blood cell separation membrane412 is no more necessary but, instead, a protein removing membrane isused preferably.

Like the rate of change of the DC component of the current between thesource 405 and the drain 406, the quantity of change of the DC componentof the current between the source 405 and the drain 406 in apredetermined period depends on the concentration of the measuredsubstance. Accordingly, the concentration of the measured substance canbe obtained by measuring the quantity of change of the DC component ofthe current between the source 405 and the drain 406 before injection ofthe sample solution or just after the injection of the sample solutionand at a predetermined period after the injection of the samplesolution. Further, in a case where the reproducibility for the DCcomponent of the current between the source 405 and the drain 406 beforeinjection of the sample solution is higher than the accuracy requiredfor the measurement, the concentration of the measured substance can beobtained by measuring the DC component of the current between the source405 and the drain 406 at a predetermined time after the injection of thesample solution.

It is preferred that the redox compound 415 is previously in a reducedstate in a case of measuring an oxidation substance such as hydrogenperoxide. For providing the reduced state, the substance is treated witha reducing agent such as potassium ferrocyanide, sodium thiosulfate, orDithiothreitol (DTT). By providing the reduced state, it is possible tostabilize the potential before the injection of the sample solution and,further, improve the sensitivity at a low concentration to extend adynamic range. In the same manner, in a case of measuring a reducingsubstance such as a thiol compound, it is preferred that the substanceis previously in an oxidized state. For providing an oxidized state, thesubstance is treated with an oxidizing agent such as potassiumferricyanide, potassium permanganate, or hydrogen peroxide. By applyingthe oxidizing treatment, it is possible to stabilize the potentialbefore injection of the sample solution and, further, improve thesensitivity at a low concentration to extend a dynamic range.

In a case where the measured substance is oxidized or reduced by theaction of the enzyme 414 and, as a result, the oxidation and reductionstate of the redox compound 415 changes, the oxidation and reductionstate of the redox compound of the redox compound 415 may be changed byway of hydrogen peroxide as described above, by way of another redoxcompound as shown in FIG. 2, or directly by the oxidation reduction forthe measured substance not by way of any redox compound.

For the insulative molecules 416, an alkane thiol is used preferably. Bythe use of the alkane thiol, a highly insulating monolayer can be formedeasily on the surface of a noble metal such as gold or silver. With aview point of an insulative property, the alkyl chain in the alkanethiolhas a carbon chain of 6 or more.

For the quasi-reference electrode 408, a silver chloride electrode or anelectrode coated with a silver/silver chloride paste is preferred.Further, the entire apparatus can be made smaller by forming thefield-effect transistor 404 and the quasi-reference electrode 408 on oneidentical substrate. The gel 409 containing a solution for referencevoltage is used for keeping the fluctuation of the potential of thequasi-reference electrode 408 smaller than the accuracy required formeasurement during measurement. For example, in a case of using thesilver-silver chloride electrode for the quasi-reference electrode 408,a gel containing an aqueous solution of potassium chloride is usedpreferably. Further, instead of the gel 409 containing the solution forreference voltage, a hydrophilic fluoro resin film or a porous film canbe used. In a case where the fluctuation of the potential of thequasi-reference electrode 408 is less than the accuracy required for themeasurement without using the gel 409 containing the solution forreference voltage, such gel 409 containing the solution for referencepotential may not be used.

Instead of the gold electrode 417, an electrode comprising other noblemetal such as silver may also be used.

The power source 407 is preferably a power source containing an ACcomponent. By superimposing an AC voltage at 1 KHz or higher to the DCcomponent, stabilization of the surface potential of the gold electrode417 can be expected.

For oxidation and reduction of the measured substance by the enzyme 414,an enzyme for the pretreatment of the measured substance or a substrateneeded for redox reaction may sometimes be used.

As the enzyme 414 in accordance with the measured substance, thoseexemplified in Table 1 and Table 2 can be used.

FIG. 5 is a block diagram showing an example of a potentiometric enzymesensor using an FET sensor according to the invention capable ofmeasuring plural targets simultaneously. The measuring system of thisembodiment includes a measuring station 501, a signal processing circuit502, and a data processing apparatus 503. The measuring station 501includes an insulated gate field-effect transistor 504, aquasi-reference electrode 512, a sample solution injector 514 forsupplying a sample solution containing a measured substance and ameasuring cell 515. The insulated gate field-effect transistor 504 has aplurality of combinations each comprising a source 505, a drain 507, agold electrode 506 electrically connected with the gate, and a source508, a drain 510, and a gold electrode 509 electrically connected withthe gate. In the measuring cell 515, there are placed a blood cellseparation membrane 516, a interfering substances separation membrane517, the quasi-reference electrode 512, and a gel 513 containing asolution for reference potential, and an enzyme 518 and a redox compound519 immobilized through insulative molecules 520 to the gold electrode506, and an enzyme 521, and a redox compound 522 immobilized through aninsulative molecules 523 to the gold electrode 509, corresponding to therespective gold electrodes 506, 509. The respective gold electrodes 506,509, the enzymes 518, 521, and the redox compounds 519, 521 immobilizedto the gold electrodes 506, 509 through the insulative molecules 520,523 are separated by a separation wall 524. In this case, while anoxidase for oxidation and reduction is used as the enzyme, any enzymethat produces an oxidized substance or a reduced substance by anenzymatic reaction can be used with no trouble. For example, acetylcholinesterase that hydrolyzes acetylthiocholin to produce a thiolcompound, which is a reduced substance, can also be used.

The measuring procedures are as described below. A predetermined voltageis applied from the power source 511. A sample solution such as a bloodis injected by using a sample solution injector 514 in the measuringcell 515. Blood cells in the sample solution are removed by the bloodcell separation membrane 516 and interfering substances giving an effecton the surface potential such as ascorbic acid in the sample solutionare removed by the interfering substances separation membrane 517. Theliquid permeating the separation membranes dissolves the enzymes 518,521. The measured substance in the sample solution is oxidized by theenzymatic reaction and, along with the reaction, the dissolved oxygen inthe sample solution is reduced to hydrogen peroxide. The hydrogenperoxide produced by the enzyme 518, 521 oxidizes the redox compounds519, 522. As a result, the potentials on the gold electrodes 506, 509are changed. The DC component of the current between the source 505 andthe drain 507, and between the source 508 and the drain 510 in theinsulated gate field-effect transistor 504 is measured at a real timeand recorded by the signal processing circuit 502 and the dataprocessing apparatus 503. The rate of change of the DC component of thecurrent between the source 505 and the drain 507 depends on thepotential of the gold electrode 506, the potential of the gold electrode506 depends on the rate of producing hydrogen peroxide, that is, thereaction rate of oxidation of the measured substance corresponding tothe enzyme 518, and the reaction rate of the oxidation of the measuredsubstance depends on the concentration of the measured substance.Accordingly, by measuring the rate of change of the DC component of thecurrent between the source 505 and the drain 507, a concentration of themeasured substance corresponding to the enzyme 518 can be obtained. Inthe same manner, the concentration of the measured substancecorresponding to the enzyme 521 can be obtained by measuring the rate ofchange of the DC component of the current between the source 508 and thedrain 510.

In a case of using urine as the sample solution, the urine is preferablydiluted and injected into the measuring cell 515 by using the samplesolution injector 514. In this case, the blood cell separation membrane516 is no more necessary but, instead, a protein removing membrane isused preferably.

The separation wall 524 serves to prevent dissolved enzymes 518 and 521from mixing to each other and not to cause cross talk. As the separationmembrane 524, a hydrophilic fluoro resin film or a porous film is usedpreferably. An identical effect can be obtained also by immobilizing theenzyme on a polymeric film and disposing the polymeric film on the redoxcompound instead of using the separation wall.

It is preferred that the redox compounds 519, 522 are previously in areduced state in a case of measuring an oxidation substance suchhydrogen peroxide. For providing the reduced state, the substance istreated with a reducing agent such as potassium ferrocyanide, sodiumthiosulfate, or Dithiothreitol (DTT). By providing the reduced state, itis possible to stabilize the potential before the injection of thesample solution and, further, improve the sensitivity at a lowconcentration to extend a dynamic range. In the same manner, in a caseof measuring a reducing substance such as a thiol compound, it ispreferred that the substance is previously in an oxidized state. Forproviding an oxidized state, the substance is treated with an oxidizingagent such as potassium ferricyanide, potassium permanganate, orhydrogen peroxide. By applying the oxidizing treatment, it is possibleto stabilize the potential before injection of the sample solution and,further, improve the sensitivity at a low concentration to extend adynamic range.

In a case where the measured substance is oxidized or reduced by theaction of the enzyme 518, 521 and, as a result, the oxidation andreduction state of the redox compound 519, 522 changes, the oxidationand reduction state of the redox compound of the redox compound 519, 522may be changed by way of hydrogen peroxide as described above, by way ofanother redox compound as shown in FIG. 2, or directly by the oxidationreduction for the measured substance not by way of any redox compound.

For the insulative molecules 520, 523, an alkanethiol is usedpreferably. By the use of the alkanethiol, a highly insulating monolayercan be formed easily on the surface of a noble metal such as gold orsilver. With a view point of an insulative property, the alkyl chain inthe alkanethiol has a carbon chain of 6 or more.

For the quasi-reference electrode 512, a silver chloride electrode or anelectrode coated with a silver/silver chloride paste is preferred.Further, the entire apparatus can be made smaller by forming thefield-effect transistor 504 and the quasi-reference electrode 512 on oneidentical substrate. The gel 513 containing a solution for referencevoltage is used for keeping the fluctuation of the potential of thequasi-reference electrode 512 less than the accuracy required formeasurement during measurement. For example, in a case of using thesilver-silver chloride electrode for the quasi-reference electrode 512,a gel containing an aqueous solution of potassium chloride is usedpreferably. Further, instead of the gel 513 containing the solution forreference voltage, a hydrophilic fluoro resin film or a porous film canbe used. In a case where the fluctuation of the potential of thequasi-reference electrode 512 is less than the accuracy required for themeasurement without using the gel 513 containing the solution forreference voltage, such gel 513 containing the solution for referencepotential may not be used.

Instead of the gold electrodes 506, 509, an electrode comprising othernoble metal such as silver may also be used.

The power source 511 is preferably a power source containing an ACcomponent. By superimposing an AC voltage at 1 KHz or higher to the DCcomponent, stabilization of the surface potential of the gold electrodes506, 509 can be expected.

For oxidation and reduction of the measured substance by the enzymes518, 521, an enzyme for the pretreatment of the measured substance or asubstrate needed for redox reaction may sometimes be used.

As the enzymes 518, 521 in accordance with the measured substance, thoseexemplified in Table 1 and Table 2 can be used.

In this embodiment, an apparatus having two sets of enzymes, goldelectrodes, etc. capable of measuring two targets simultaneously hasbeen described but an apparatus capable of measuring more than threetargets simultaneously can also be constructed in the same manner.

FIG. 6 is a view showing an example of a structure for an analyticalelement used in a potentiometric enzyme sensor using an FET sensoraccording to the invention. FIGS. 6A and 6B respectively show a crosssectional structure and a planer structure. In an insulated gatefield-effect transistor 601, a source 602, a drain 603, and a gateinsulator 604 are formed on the surface of a silicon substrate, and agold electrode 605 is arranged. The gold electrode 605 and the gate 606of the insulated gate field-effect transistor are connected through aconductive wiring 607. The insulated gate field-effect transistor is,preferably, a metal-oxide semiconductor field-effect transistor using asilicon oxide as an insulative film, but a thin film transistor (TFT)may also be used with no problem. By adopting this structure, the goldelectrode 605 can be formed at an arbitrary place to an arbitrary size,and the capacity of the measuring cell can be varied in accordance withthe amount of the sample solution as a measured substance. The insulatedgate field-effect transistor used in the invention is a depletion typeFET having an insulation layer using SiO₂ (thickness: 17.5 nm) and thegold electrode is prepared to a size of 400 μm×400 μm. Since an aqueoussolution is used for the usual measurement, the device has to operate ina solution. In a case of measurement in the solution, it is necessarythat the device operates in an electrode potential range from −0.5 to0.5 V causing less electrochemical reaction. For this purpose, in thisembodiment, the threshold voltage of FET is set to about −0.5V bycontrolling parameters for manufacturing the depletion type n-channelFET, that is, the parameters for ion implantation to control thethreshold voltage (Vt). Instead of the gold electrode, an electrodecomprising other noble metal such as silver may also be used. A devicehaving two or more YET sensors on one identical substrate is prepared byforming the FET sensor in FIG. 6 in plurality on one identicalsubstrate. Cross talk between the FET sensors causing a problem in thiscase can be mitigated by adopting an SOI (Silicon on Insulator)structure.

The effect of immobilizing the redox compound on the surface of theworking electrode through the insulative molecules according to theinvention is to be described with reference to other example. FIG. 7Ashows an evaluation system in a case of using an unmodified workingelectrode and FIG. 7B shows an evaluation system in a case ofimmobilizing a redox compound to the surface of a working electrodethrough insulative molecules. The evaluation system in a case of usingthe unmodified working electrode includes a potentiometric measuringdevice 701, a reference electrode 702, and a measuring cell 703. In themeasuring cell 703, there are placed a measuring solution 704, a goldelectrode 705, and a reference electrode 702. The evaluation system in acase of immobilizing a redox compound through insulative molecules tothe surface of a working electrode includes a potentiometric measuringdevice 711, a reference electrode 712, and a measuring cell 713. In themeasuring cell 713, there are placed a measuring solution 716, a goldelectrode 717 in which a redox compound 714 is immobilized throughinsulative molecules 715, and the reference electrode 712.

FIG. 8 shows a potential difference between the reference electrode andthe gold electrode against the logarithm of a redox compoundconcentration ratio in the measuring solution (logarithm for the ratioof potassium ferricyanide concentration and potassium ferrocyanideconcentration). In FIG. 8, open triangles (A) show the result ofmeasurement using the evaluation system of FIG. 7A and open circles inFIG. 8 shows the result of measurement using the evaluation system ofFIG. 7B. For the reference electrodes 702, 712, a silver chloridereference electrode using an aqueous solution of saturated potassiumchloride as the internal solution is used. For the redox compound 714immobilized through insulative molecules 715,11-ferrocenyl-1-undecanethiol (11-FUT) was used. For the measuringsolutions 704, 716, a 0.1M aqueous solution of sodium sulfate containingpotassium ferricyanide and potassium ferrocyanide at a totalconcentration of 10 μM was used. The measuring temperature was 25° C.Within a range of the logarithm of the redox compound concentrationratio from −1 to 3, both the open triangles and the open circles can bewell fitted with a line of 58 mV/decade and it can be seen that thepotential is generated in accordance with the Nernst's equation.However, the open triangles deviate from the line at the logarithm forthe redox compound concentration ratio of −2 or less or 4 or more andthe range of the potential fitted with the line is 134 to 409 mV, whichcorresponds to the concentration range of 4.7 digits. On the other hand,for the open circles, the range of the voltage fitted with the line isfrom 80 to 442 mV, which corresponds to the concentration range of 6.2digits. That is, by immobilizing the redox compound through theinsulative molecules on the surface of the working electrode, thedynamic range was improved by 1.5 digits.

Without immobilization of the insulative molecules to the surface of theworking electrode, the insulative property between the working electrodeand the measuring solution is low. Accordingly, due to the effect of theleak current on the electrode surface, minute change of theconcentration of the measured substance cannot be detected. On the otherhand, by immobilizing the redox compound through the insulativemolecules on the surface of the working electrode, insulative propertybetween the working electrode and the measuring solution is increased todecrease the leak current on the surface of the electrode, and a furtherminute change of the measured substance can be detected. In this case,by immobilizing the redox compound through the insulative molecules ofan identical length on the surface of the working electrode, the workingelectrode can obtain insulative property and change of state of theimmobilized redox compound can give an effect on the surface potentialof the working electrode uniformly.

The effect of immobilizing the redox compound through the insulativemolecules to the surface of the working electrode in the invention is tobe described with reference to other embodiment. FIG. 9 showsvoltammograms of a gold electrode in which an unmodified gold electrodeand insulative molecules are immobilized. For the potentiostat, anelectrochemical analyzer ALS Model 611B was used. As the referenceelectrode, a silver chloride reference electrode using a saturatedaqueous solution of potassium chloride as the internal solution is used.As the counter electrode, a platinum wire was used. As the solution, a0.1 M aqueous solution of sodium sulfate is used. In the voltamogram,the difference of current value depending on the sweeping directionshows the capacitance of the surface of the electrode and the slope ofthe current value against the applied potential shows the resistance ofthe electrode surface. Compared with the unmodified gold electrode, in agold electrode on which 11-hydroxy-1-hexanthiol (11-HUT) was immobilizedas the insulative molecules, the slope of the current value against theapplication potential was small. This means that the insulative propertyat the surface of the electrode is increased by immobilization of theinsulative molecules on the electrode surface. Further, compared withthe unmodified gold electrode, in the gold electrode immobilizing 11-HUTas the insulative molecules, the absolute value of the current wassmall. This is because a layer of about one molecule referred to as anelectric double layer is present on the surface of the electrode in theunmodified gold electrode and the surface has such a large capacitanceas 14 μF/cm², whereas the insulative molecules are immobilized to theelectrode surface thereby forming an insulation layer of about 2 nm asthe length of insulative molecules, and the capacitance is reduced to2.3 μF/cm². By immobilizing the insulative molecules to the surface ofthe electrode, the resistance value on the surface of the electrode isincreased and the capacitance is decreased. As a result, the leakcurrent is decreased and, further, charging current is also decreased,so that further miner change of the concentration of the redox compoundcan be recognized.

The effect of immobilizing the redox compound through the insulativemolecules on the surface of the working electrode according to theinvention is to be described with reference to other example. FIG. 10shows an evaluation system in a case of immobilizing the redox compoundthrough insulative molecules on the surface of the working electrode.The evaluation system includes a potentiometric device 801, a referenceelectrode 802, a sample solution injector 803 for supplying a samplesolution containing measured substance, and a measuring cell 804. In themeasuring solution 807 in the measuring cell 804, a gold electrode 808and a reference electrode 802 on which the redox compound 805 isimmobilized through insulative molecules 806 are arranged.

FIG. 11 shows the time course of the potential difference between thereference electrode and the gold electrode upon injecting a measuredsubstance. For the reference electrode 802, a silver chloride referenceelectrode using an saturated aqueous solution of potassium chloride asthe internal solution is used. For the redox compound 805 immobilizedthrough the insulative molecules 806, (a) 6-ferrocenyl-1-hexanethiol,(b) 8-ferrocenyl-octanethiol, and (c) 11-FUT are used. For the samplesolution in the sample solution injector, an aqueous solution ofpotassium ferrocyanide was used. For the measuring solution 807, a 0.1Maqueous solution of sodium sulfate was used. The abscissa in FIG. 11shows the time with the instance of sample injection as 0, and theordinate shows the potential between the reference electrode and thegold electrode which is normalized with the potential just before theinjection of the sample solution as 1 and the potential 100 sec afterthe injection as 0. Assuming the time till the potential changes to 0.1by the injection of the sample solution, that is, the time required forthe 90% potential change as a relaxation time, the relaxation time wasin the order of (a)>(b)>(c). That is, as the length of the insulativemolecules 806 is longer, the relaxation time was shorter. As the lengthof the insulative molecules 806 is longer, the insulative propertybetween the working electrode and the measuring solution is higher. Thatis, as the insulative property is higher, the response speed is faster.

FIG. 12 shows the result of measuring hydrogen peroxide by using apotentiometric sensor in which the redox compound is immobilized throughthe insulative molecules on the working electrode according to theinvention. In this example, measurement is conducted by the constitutionof the apparatus shown in FIG. 7B. 11-FUT was used for the redoxcompound immobilized through the insulative molecules on the goldelectrode, a 0.1M aqueous solution of sodium sulfate is used for themeasuring solution, and a silver chloride electrode using saturatedpotassium chloride as the internal solution is used for the referenceelectrode. The abscissa in FIG. 12 shows the concentration of hydrogenperoxide and the ordinate represents the potential. This result showsthat a potential in accordance with the concentration of hydrogenperoxide is generated. This is because the redox compound immobilized onthe surface of the gold electrode is oxidized by hydrogen peroxide.

FIG. 13 shows the result of measuring a glucose concentration by usingthe potentiometric enzyme sensor according to the invention. In thisembodiment, measurement was conducted by the constitution of theapparatus shown in FIG. 2. Generally, in a case where glucose is at ahigh concentration, since the concentration of dissolved oxygen in theblood (partial pressure) is insufficient to restrict the reaction,potassium ferricyanide is used as another redox compound instead ofoxygen. 11-FUT is used for the redox compound immobilized through theinsulative molecules, a 0.1M phosphate buffer solution (PBS) at pH 7.4is used for the measuring solution, a glucose oxidase was used as theenzyme, potassium ferrocyanide was used as a water soluble redoxcompound, and a silver chloride electrode using saturated potassiumchloride as the internal solution is used for the reference electrode.Aqueous solutions of glucose at 1, 10, 100, 1,000 mg/dl are used for thesample solution. The abscissa shows a glucose concentration and theordinate shows a potential difference before injection and 100 sec afterthe injection of the measured substance. This result shows that apotential in accordance with the glucose concentration is generated.This is because the reaction of glucose and potassium ferricyanidecatalyzed by the glucose oxidase produces gluconolactone and potassiumferrocyanide, and ferrocene immobilized on the gold electrode is reducedby produced potassium ferrocyanide.

FIG. 14 shows a result of measuring cholesterol by using thepotentiometric enzyme sensor according to the invention. In thisembodiment, measurement was conducted by the constitution of apparatusshown in FIG. 1. 11-FUT was used for the redox compound immobilizedthrough the insulative molecules. A silver chloride electrode usingsaturated potassium chloride as the internal solution was used for thereference electrode. As the measuring solution, a mixed solution whichcontains 20 μl of 0.046 to 100 mg/dl cholesterol solution (dissolved in2% aqueous solution of Triton X-100), 10 μl of 3 mg/ml NAD solution(dissolved in 0.3 M aqueous solution of Tris-HCl (pH 8.5)), 3 μl of 100mM aqueous solution of potassium ferricyanide, and 1 μl of 100 U/mlaqueous solution of diaphorase was used. 1 μl of 10 mg/ml cholesteroldehydrogenase was injected by using the sample solution injector, andthe potential difference between the reference electrode and the goldelectrode 600 sec after the injection was plotted in FIG. 14. A slopesensitivity of 60 my was obtained to the cholesterol concentration(abscissa), and it can be seen that a potential in accordance with thecholesterol concentration is generated in a range from 1 to 100 mg/dl.In this measurement, as a result of the occurrence for the reaction:cholesterol+NAD→cholestenone+NADH catalyzed by the cholesteroldehydrogenase, the reaction: NADH+ferricyan→NAD+ferrocyan catalyzed bydiaphorase, and the reaction: ferrocyan+ferrocene (oxidationform)→ferricyan+ferrocene (reduced form) on the surface of the goldelectrode, the surface potential of the gold electrode was changed inaccordance with the cholesterol concentration. By using this principle,the cholesterol concentration can be measured as the potential change.

FIG. 15 shows the result of measuring Fru-Val-His peptide obtained bytreating hemoglobin A_(lc) with a protease enzyme by using thepotentiometric enzyme sensor according to the invention. In thisembodiment, measurement was conducted by the constitution of apparatusshown in FIG. 2. 11-FUT was used for the redox compound immobilizedthrough the insulative molecules, a PBS solution was used for themeasuring solution, Fluctosyl-peptide Oxidase (FPDX) was used as theenzyme, 1 mM potassium ferrocyanide was used for the water soluble redoxcompound, and a silver chloride electrode using saturated potassiumchloride as the internal solution was used for the reference electrode.An aqueous solution of Fru-Val-His peptide substrate was used for thesample solution. The abscissa shows the final concentration of thesubstrate and the ordinate shows the potential difference beforeinjection of the measured substance and 600 sec after injection. Thisresult shows that a potential in accordance with the concentration ofthe substrate is generated. The slope sensitivity in this case was about60 mV and it can be seen that the potential is generated in accordancewith Nernst's equation.

FIG. 16 shows the result of measuring the cholinesterase activity byusing the potentiometric sensor in which the redox compound wasimmobilized through the insulative molecules on the working electrodeaccording to the invention. In this example, measurement was conductedwith the constitution of apparatus shown in FIG. 7B. 11-FUT was used forthe redox compound immobilized through the insulative molecules on thegold electrode, PBS (phosphate buffered saline) containing acetylthiocholine at a concentration of 1 mg/ml (5.06 mM) was used for themeasuring solution, and a silver chloride electrode using saturatedpotassium chloride as the internal solution was used for the referenceelectrode. An acetylcholinesterase solution was used for the samplesolution. When the sample solution is injected into the measuringsolution, acetylthiocholine is decomposed into thiocholine and aceticacid by acetylcholinesterase in the sample solution. When thiocholine isproduced, since thiocholine acts as a reducing agent to ferroceneimmobilized on the working electrode, the surface potential of theworking electrode is lowered. On the other hand, since acetylthiocholinehas no thiol groups at the terminal, it does not act as the reducingagent and gives no effect on the surface potential of the workingelectrode. Accordingly, the potential was lowered in accordance with theactivity of acetylcholinesterase in the sample solution. FIG. 16 showsthe activity of acetylcholinesterase in the sample solution on theabscissa and the rate of potential change just after the injection ofthe sample solution on the ordinate. The rate of potential change inproportion with the activity of acetylcholinesterase was observed. Fromthe result, the concentration of acetylcholinesterase can be determinedbased on the rate of potential change. In addition to the rate ofpotential change, the quantity of change of the potential difference canalso be used and since the quantity of change of the potentialdifference in proportion to the logarithm of the cholinesterase activityis obtained in this case, a wider dynamic range can be obtained.

As other application example, organophosphate pesticides that inhibitthe acetylcholinesterase activity can be measured. A solution containinga sufficient amount of acetylcholine was used for the measuring solutionand a solution containing a predetermined amount of acetylcholinesteraseand a measured substance is used for the sample solution. In a casewhere a pesticides that inhibits acetylcholinesterase activity containedin the sample solution, the decomposing rate of acetylthiocholine byacetylcholinesterase is lowered. Accordingly, the concentration of theorganophosphate pesticides as the acetylcholinesterase inhibitorcontained in the sample solution can be measured based on thedecomposing rate of acetylcholine, that is, the rate of the potentialchange.

Also in a case of using an unmodified gold electrode instead of the goldelectrode on which the redox compound is immobilized through theinsulative molecules, the potential change due to the bonding ofthiocholine to the surface of the gold electrode can be measured.However, the gold electrode is disposed after use in this case. On theother hand, in a case of using the gold electrode on which the redoxcompound is immobilized through the insulative molecules according tothe invention, since thiocholine is not bonded to the surface of thegold electrode, the gold electrode can be used for several times bywashing to regenere. For regeneration, potassium ferricyanide orhydrogen peroxide as an oxidizing substance is used preferably.

FIG. 17 shows the result of repetitively measuring the concentration of4-hydroxy-1-butanthiol (4-HBT) as a thiol compound by using an FETsensor in which the redox compound is immobilized through the insulativemolecules to the working electrode according to the invention. 11-FUTwas used for the redox compound immobilized through the insulativemolecules on the gold electrode, a 4-HBT solution (dissolved in PBS) wasused for the measuring solution, and a silver chloride electrode usingsaturated potassium chloride as the internal solution was used for thereference electrode. After washing with 5% aqueous hydrogen peroxide forthree times before measurement, measurement was conducted in the orderof 4-HBT concentration at 10⁻⁷M, 10⁻⁶M, 10⁻⁵M, 10⁻⁴M, 10⁻³M, 10⁻²M, and10⁻¹M. The result is shown in FIG. 17A (1st time). As the 4-HBTconcentration increased, a drain current value was decreased. This isbecause ferrocene immobilized on the surface of the gold electrode isreduced by 4-HBT and the potential on the surface of the gold electrodewas lowered. Again, after washing with 5% aqueous hydrogen peroxide forthree times, the concentration of 4-HBT was changed and measured in thesame manner. The result is shown in FIG. 17A (2nd time). Like (1sttime), a drain current value in accordance with the 4-HBT concentrationwas observed. In the same manner, when (3rd) and (4th) were measured,same results were also obtained. The drain current values were convertedinto the surface potential of the gold electrode based on the gatevoltage (VG)-drain current (ID) characteristic of the FET measuredseparately, and the decrement of the voltage (−ΔV) was plotted with10⁻⁷M as the reference (FIG. 17B). In any measurement, a potentialchange depending on the 4-HBT concentration was observed, theimmobilized redox compound was in an oxidized state by washing with anoxidizing agent such as hydrogen peroxide and it was confirmed that thethiol compound can be measured repetitively. Also in a case of using theunmodified gold electrode instead of the gold electrode on which theredox compound was immobilized through the insulative molecules, theconcentration of the thiol compound can be measured due to the potentialchange by the bonding of the thiol compound to the surface of the goldelectrode. However, in this case, the gold electrode is disposed afteruse. On the other hand, by the use of the gold electrode on which theredox compound was immobilized through the insulative molecules of theinvention, since the thiol compound is not bonded to the surface of thegold electrode, the gold electrode can be used for several times bywashing and regeneration.

FIG. 18 is a block diagram showing an example of an immunoassay systemusing an FET sensor according to the invention. The analytic systemincludes a measuring station 901, a signal processing circuit 902, adata processing apparatus 903, and a reactor 904 for a thiol compoundproducing reaction. In the measuring station 901, there are placed aninsulated gate field-effect transistor 905, a reference electrode 906, apower source 907 for applying a high frequency voltage to the referenceelectrode 906, and a thiol compound solution injector 908 for supplyingthe solution in the reactor 904. A gold electrode 913 formed on theinsulated gate field-effect transistor 905 and a reference electrode 906are provided in a reaction solution 910 in a measuring cell 909. A redoxcompound 911 is immobilized through an insulative molecules 912 on thegold electrode. In the reactor 904 for the thiol compound producingreaction, an antibody 916 is immobilized on an antibody immobilizingplate 917. The antibody 916 may be directly immobilized on the inside ofthe reactor 904.

The measuring procedures are as described below. A sample solution wasinjected to the reactor 904 for the thiol compound producing reaction,to bind an antigen and the antibody 916 in the sample solution. After apredetermined time, an enzyme labeled antibody solution is injected intothe reactor 904 to cause antigen-antibody reaction and form anantibody-antigen-enzyme labeled antibody. Then, the bound enzyme labeledantibody and the free enzyme labeled antibody are separated by exchangeof the solution in the reactor 904 and washing of the reactor 904. Afterexchange of solution and washing in the reactor 904, when the substratefor the labeling enzyme is injected, the substrate is decomposed by theenzyme to produce a thiol compound. After reaction for a predeterminedtime, the produced thiol compound is introduced by using a thiolcompound solution injector 908 to the reaction solution 910 in themeasuring cell 909. The thiol compound introduced to the reactionsolution 910 in the measuring cell 909 reduces the redox compound 911immobilized on the gold electrode 913 formed on the insulated gatefield-effect transistor 905. As a result, the potential on the goldelectrode 913 changes. The current between a source 914 and a drain 915in the insulated gate field-effect transistor 905 that changes beforeand after the injection of the produced thiol compound is measured onreal time and recorded by the signal processing circuit 902 and the dataprocessing apparatus 903. Since the potential on the gold electrode 913is determined by the ratio of the oxidized state and the reduced stateof the redox compound 911, a potential depending on the concentration ofthe thiol compound is generated to the gold electrode 913. Accordingly,the amount of the bound labeling enzyme, that is, the amount of theantigen in the sample solution can be obtained by measuring the value ofthe current between the source 914 and the drain 915 that changes withthe potential of the gold electrode 913.

The enzyme immunoassay using the apparatus of the invention is to bedescribed below. In this embodiment, the amount of the antigen wasmeasured indirectly through an enzyme labeled to the antibody by using asandwich method generally used in immunoassay. After reaction betweenthe antibody immobilized previously on the plate and the antigen in thesample, an enzyme labeled antibody was added to form anantibody-antigen-enzyme labeled antibody. Then, the bound enzyme labeledantibody, and the free enzyme labeled antibody and the free antigen wereseparated, and the thiol compound as a product of a cyclic reactionbetween enzyme of the bound enzyme labeled antibody and the substratewas measured by the FET sensor. The samples and the reagents used inthis embodiment are shown below. Immobilized antibody: Interleukin 1a(IL-1β) antibody

-   Sample: Human plasma-   Measured substance: IL-1β-   Enzyme labeled antibody: Acetylcholinesterase (AchE): IL-1β Fab′    Conjugate-   Substrate: 2.5 mM Acetylthiocholine-   Reaction solution: PBS

The reaction conditions and reagent concentrations used herein are mereexamples and can be changed properly depending on the constitution ofapparatus and the measured substance.

The measuring procedures are as described below. At first, 100 μL of asample solution (Human plasma) and 100 μL of an enzyme labeled antibody(AchE: IL-1β Fab′ Conjugate) were added to a well of a plate on whichthe IL-1β antibody was immobilized, the plate was covered with a plasticfilm and they were reacted at 4° C. for overnight. Then, the solution inthe well of the plate was discarded and the plate was washed with awashing buffer for 5 to 6 times. An acetylthiocholine solution as asubstrate solution for acetylcholine esterase was added to each of thewells and reacted for about 20 min. The reaction solution containing thethiol compound produced by the reaction was introduced into a reactioncell in which the FET sensor was dipped, and the potential change on thegold electrode due to the redox reaction between the thiol compound andthe redox compound immobilized on the gold electrode was measuredthereby obtaining a concentration of the produced thiol compound. Sincethe concentration of the produced thiol compound is in proportion withthe enzyme concentration of the antibody-antigen-enzyme labeledantibody, the amount of antigen can be determined quantitatively.

Upon measurement by the FET sensor, 11-FUT was used for the redoxcompound immobilized through the insulative molecules, and an Ag/AgClreference electrode was used as the reference electrode. An AC voltagewith a center voltage of 100 mV and an amplitude voltage of 100 mV, andat a frequency of 1 MHz was applied to the reference electrode. 1.0V wasapplied between the source and the drain, and the drain current value 1min after the start of the measurement was recorded. The procedures wereconducted for IL-1β as the antigen at concentrations of 10, 30, 100,300, 1,000, 3,000, and 5,000 pg/ml orderly. Then, the FET sensor waswashed with 5% aqueous hydrogen peroxide, and the concentration of IL-1βwas measured again. FIG. 19A shows the result of the measurementconducted for three times. In each of measurements, a correlation thatthe drain current value decreases as the IL-1β concentration increaseswas obtained and a similar result was obtained in any measurement. Theresult was converted to the surface potential of the gold electrode byusing the FET gate voltage (VG)-drain current (ID) characteristicsmeasured separately, and the decrement of the potential (−ΔV) wasplotted with 10 pg/ml for the IL-1β concentration as the standard (FIG.19B). In any measurement, the potential change depending on IL-1βconcentration was observed, and it was confirmed that the immobilizedredox compound was in the oxidized state by washing with the oxidizingagent such as hydrogen peroxide and the concentration of the thiolcompound, that is, the antigen could be measured repetitively.

What is claimed is:
 1. A potentiometric sensor comprising: a vessel towhich a measuring solution containing an object for measurement isintroduced, a working electrode in contact with the measuring solutionin the vessel, a reference electrode in contact with the measuringsolution in the vessel, and a potentiometer connected to the workingelectrode and the reference electrode for measuring a potentialdifference between the working electrode and the reference electrode, anenzyme in the measuring solution, wherein the enzyme is not immobilizedon the working electrode or the reference electrode, and the enzyme isconfigured to produce an oxidized substance or a reduced substance formeasurement, wherein a redox compound is immobilized through insulativemolecules on the working electrode.
 2. A potentiometric sensorcomprising: a vessel to which a measuring solution containing an objectfor measurement is introduced, a working electrode in contact with themeasuring solution in the vessel, a reference electrode in contact withthe measuring solution in the vessel, and a potentiometer connected tothe working electrode and the reference electrode for measuring apotential difference between the working electrode and the referenceelectrode, wherein a redox compound is immobilized through insulativemolecules on the working electrode, and wherein an enzyme configured toproduce an oxidized substance or a reduced substance for measurement iscontained in the vessel.
 3. The potentiometric sensor according to claim2, wherein the redox compound is a ferrocene derivative.
 4. Thepotentiometric sensor according to claim 2, wherein the insulativemolecules comprise a carbon chain.
 5. The potentiometric sensoraccording to claim 2, wherein the working electrode is formed of carbonor a noble metal.
 6. The potentiometric sensor according to claim 2,wherein the object for measurement is an ingredient contained in abiological sample.
 7. The potentiometric sensor according to claim 6,wherein the biological sample is blood or urine.